Team:Tianjin

From 2009.igem.org

1,Cyanobacteria converter.
Inspired by several features of cyanobacteria, which is low-grade, fast-growing, photosynthetic and easy to operate. We aim to construct an pathway in cyanobacteria so that when it is carrying photosynthesis，carbon dioxide can be transferred into target production, ethanol.

2,Another project is taking use of Yeast Gal4 system which is aimed at Microcystins(MCs, induce liver cancer) detection in waters.

Why we design a detector?

The threat of MCs in seas, rivers, lakes and Reservoirs has become more and more severe worldwide due to eutrophication in pulltion waters. As we know, there are far more nitrogen and Phosphor elements in polluted waters, which is called eutrophication, resulting in algal blooms and red tides. Fishes, prowns and almost all the living creatures in these waters are killed by the high concentration of MCs that are produced by algea. It is also a severe threat to drinking water and water in daily use, for MCs induce liver cancer. What's more, the existed detecting technologies are complicated, expensive, time-consuming and require an expert to handle the devices.

Background knowledge of our project:

1. To active Gal4 promoter, two domains must come to each other close enough in the nucleus of Yeast. One is AD(Activation domain), the other is DNA-BD(DNA Binding domain). When AD and DNA-BD are close enough to each other, the Report genes transcription LacZ will be started. Then we use X-gal to detect whether LacZ is expressed( LacZ could turn the color of X-gal from white to blue).
2. According to the Toxicity mechanism, Adda binding site of MCs has a strong affinity to the lyophobic groove of PP1. Also, according to the Detoxicity mechanism, GSH could construct a covalent bond with Mdha banding site of MCs easily under the function of GST(Glutathione S-Transferase Agents which presents in the cell of Yeast)

Our design:

In our project, we connect PP1 gene after DNA-BD in the plasmid pGBKT7 and GSH gene after AD in the plasmid pGADT7, and then we transform these two plasmids into Yeast(AH109) so that it can express protein DNA-BD-PP1 and AD-GSH. At last our hero MCs enters. It will connect with AD-GSH which has a system can lead it to the nucleus of yeast, and then AD-GSH-MCs comes into the core to connect with DNA-BD-PP1 on chromosome. Expression of both fusion proteins in yeast and interaction between bait and prey indeed reconstituted a functional Gal4 transcription factor from the two separate polypeptides. Gal4 then recruited RNA polymerase II, leading to transcription of a GAL1-lacZ fusion gene. This reporter gene encodes the enzyme beta-galactosidase which labels the yeast cell when using a colorimetric substrate